Viruses for the treatment of cellular proliferative disorders

ABSTRACT

Methods for treating cell proliferative disorders by administering virus to proliferating cells having an activated Ras-pathway are disclosed. The virus is administered so that it ultimately directly contacts proliferating cells having an activated Ras-pathway. Proliferative disorders include but are not limited to neoplasms. The virus is selected from modified adenovirus, modified HSV, modified vaccinia virus and modified parapoxvirus orf virus. Also disclosed are methods for treating cell proliferative disorders by further administering an immunosuppressive agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/418,290,filed on Apr. 18, 2003, which is a continuation of application Ser. No.09/708,663, filed on Nov. 9, 2000, now U.S. Pat. No. 6,596,268 whichclaims the benefit of U.S. Provisional Application No. 60/164,878, filedNov. 12, 1999. The disclosure of each of the above applications isincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention pertains to methods for treating cellularproliferative disorders in a mammal that are mediated by Ras-activationusing mutant viruses.

REFERENCES

The following publications, patent applications and patents are cited inthis application:

-   1. Beattie, E. et al., Virology (1991) 183:419-422-   2. Black, T. L., et al., J. Virol. (1993) 67:791-800-   3. Chang, H. W. and Jacobs, B. L. Virology (1993) 194:537-547-   4. Chong, K. L. et al., EMBO J. (1992) 11:1553-1562-   5. Davies, M. V. et al., JBC (1991) 266:14714-14720-   6. Davies, M. V. et al., J. Virology (1993) 67:1688-1692-   7. Jagus, R. and Gray M. M. Biochimie (1994) 76:779-791-   8. Katze, M. G. et al., EMBO J(1987) 6:689-697-   9. Katze M. G. et al., Trends in Microbiology (1995) 3:75-78-   10. Lee, T. G. et al., MCB (1994) 14:2331-2342-   11. Mundshau, L. J. and Faller, D. V., JBC (1992) 267:23092-23098-   12. Mundshau, L. J. and Faller, D. V., Biochimie (1994) 76:792-800-   13. Nanduri, S. EMBO J. (1998) 17:5458-5465-   14. Proud, D. G. Trends in Biochemical Sciences, (1995) 20:241-246-   15. Redpath, N. T. and Proud, D. G. Biochimica et Biophysica    Acta, (1994) 1220:147-162-   16. Strong, J. E. et al., EMBO (1998) 17:3351-3362-   17. Williams, B. R., Biochemical Society Transactions (1997)    25:509-513.-   18. Wiessmuller, L. and Wittinghofer, F., Cellular Signaling (1994)    6(3):247-267-   19. Barbacid, M., A Rev. Biochem. (1987) 56:779-827-   20. Millis, N. E. et al., Cancer Res. (1995) 55:1444-   21. Chaubert, P. et al., Am. J Path. (1994) 144:767-   22. Bos, J., Cancer Res. (1989) 49:4682-   23. Levitzki A., Eur. J. Biochem. (1994) 226:1-   24. James P. W., et al., (1994) Oncogene 9:3601-   25. Lee J. M. et al., PNAS (1993) 90:5742-5746-   26. Lowe S. W. et al., Science, (1994) 266:807-810-   27. Raybaud-Diogene H. et al. J. Clin. Oncology, (1997)    15(3):1030-1038.-   28. Brooks et al., eds. “Jawetz, Melnick, & Adelberg's Medical    Microbiology,” (1998)-   29. He, B. et al, PNAS (1997) 94:843-848-   30. Haig, D. M. et al., Immunology (1998) 93:335-340-   31. Kawagishi-Kobayashi, M. et al., MCB (1997) 17:4146-4158-   32. Martuza et al., European Patent Application Publication Number    EP 0 514 603, published Nov. 25, 1992

All of the above publications, patent applications and patents areherein incorporated by reference in their entirety to the same extent asif each individual publication, patent application or patent wasspecifically and individually indicated to be incorporated by referencein its entirety.

State of the Art

Normal cell proliferation is regulated by a balance between growthpromoting proto-oncogenes and growth-constraining tumor-suppressorgenes. Tumorigenesis can be caused by genetic alterations to the genomethat result in the mutation of those cellular elements that govern theinterpretation of cellular signals, such as potentiation ofproto-oncogene activity or inactivation of tumor suppression. It isbelieved that the interpretation of these signals ultimately influencesthe growth and differentiation of a cell, and that misinterpretation ofthese signals can result in neoplastic growth (neoplasia).

Genetic alteration of the proto-oncogene Ras is believed to contributeto approximately 30% of all human tumors.^(18, 19) The role that Rasplays in the pathogenesis of human tumors is specific to the type oftumor. Activating mutations in Ras itself are found in most types ofhuman malignancies, and are highly represented in pancreatic cancer(80%), sporadic colorectal carcinomas (40-50%), human lungadenocarcinomas (15-24%), thyroid tumors (50%) and myeloid leukemia(30%).^(20, 21, 22) Ras activation is also demonstrated by upstreammitogenic signaling elements, notably by tyrosine receptor kinases(RTKs). These upstream elements, if amplified or overexpressed,ultimately result in elevated Ras activity by the signal transductionactivity of Ras. Examples of this include overexpression of PDGFR incertain forms of glioblastomas, as well as in c-erbB-2/neu in breastcancer.^(22.23.24)

Protein kinase R (“PKR”) is a serine/threonine kinase that is induced inthe presence of interferon.^(7, 9, 17) The primary cellular substrate ofthis kinase is the α subunit of the translation initiation factor eIF-2on Serine 5.^(14,15,17) Phosphorylation of eIF-2 results in a rapidinhibition of protein synthesis by preventing its participation infurther rounds of translation initiation.

Although PKR is normally inactive, it becomes rapidly activated in thepresence of double stranded RNA (dsRNA) or RNAs that exhibit extensivesecondary structures, elements that are frequently produced as theresult of viral infection. The amino-terminal of PKR contains a doublestranded RNA binding domain (dsRBD) that allows this interaction withdsRNA. Binding of PKR to dsRNA element allows PKR to undergo aconformational change that facilitates autophosphorylation andsubsequent phosphorylation of eIF-2 ⁴ Further, it appears that thecooperative binding of two PKR molecules to one dsRNA molecule isrequired to achieve activation since the addition of dsRNA to PKRresults in the dsRNA/PKR activation complex to be found in a 2:1 ratioof protein to dsRNA.¹⁷

Double-stranded RNA (dsRNA) viruses are not entirely susceptible to thehost cell PKR because they have evolved a number of different strategiesto inhibit PKR activation in response to their presence:

(1) In the case of adenovirus, a viral product, VAI RNA, is synthesizedin large amounts. These VAI RNA elements, with their extensive secondarystructure and short length inactivate PKR by acting as a competitiveinhibitor of the full length viral dsRNA.⁸ The short length of the VAIRNA elements is critical, as there is a minimum length dsRNA whichactivates PKR. PKR bound to VAI RNA is not activated;

(2) Vaccinia virus encodes two gene products, K3L and E3L todownregulate PKR with different mechanisms. The K3L gene product haslimited homology with the N-terminal region of eIF-2α and may act as apseudosubstrate for PKR.^(1,5) The E3L gene product is a dsRNA-bindingprotein and apparently functions by sequestering activatordsRNAs;^(3, 6)

(3) Herpes simplex virus (HSV) gene _(γ1)34.5 encodes the gene productinfected-cell protein 34.5 (ICP34.5) that can prevent the antiviraleffects exerted by PKR; and

(4) The parapoxvirus orf virus encodes the gene OV20.0L that is involvedin blocking PKR activity.³⁰

It has been demonstrated that in Ras transformed cells, dsRNA-mediatedactivation of PKR was blocked at the level of autophospohrylation.¹⁶

PKR is one of many cellular proteins that is induced in the presence ofinterferon (“IFN”). In normal cells, PKR is normally induced andactivated in the presence of IFN. In Ras-mediated tumor cells, however,PKR is induced in the presence of IFN but the activation of PKR isreversed or inhibited. Accordingly, Ras-mediated tumors are unable toactivate a PKR response.

It has been observed that pre-treating cells with IFN to induce thetranscription and translation of PKR prevents reovirus infection. PKRwas activated in cells that were pre-treated with IFN, suggesting thatthere may be a “quantity effect.” When the cells were not pre-treatedwith IFN, reovirus was able to replicate quickly enough such that therewas not enough time to allow sufficient PKR to be synthesized.Additionally, the PKR already present in the cell was not activated.This observation suggests that the cells are not deficient in the IFNresponse per se, since PKR is only one element of the IFN response andPKR apparently acted normally if the cells were pre-treated.

Current methods of treatment for neoplasia include surgery, chemotherapyand radiation. Surgery is typically used as the primary treatment forearly stages of cancer; however, many tumors cannot be completelyremoved by surgical means. In addition, metastatic growth of neoplasmsmay prevent complete cure of cancer by surgery. Chemotherapy involvesadministration of compounds having antitumor activity, such asalkylating agents, antimetabolites, and antitumor antibiotics. Theefficacy of chemotherapy is often limited by severe side effects,including nausea and vomiting, bone marrow depression, renal damage, andcentral nervous system depression. Radiation therapy relies on thegreater ability of normal cells, in contrast with neoplastic cells, torepair themselves after treatment with radiation. Radiotherapy cannot beused to treat many neoplasms, however, because of the sensitivity oftissue surrounding the tumor. In addition, certain tumors havedemonstrated resistance to radiotherapy and such may be dependent ononcogene or anti-oncogene status of the cell ^(25, 26, 27) Martuza etal., EP 0 514 603³², generically describes methods for selectivelykilling neoplastic cells which utilize altered viruses that are capableof replication in neoplastic cells while sparing surrounding normaltissue.

Accordingly, it has been found that viruses which have evolved certainmechanisms of preventing PKR activation are likely rendered replicationincompetent when these same mechanisms are prevented or mutated.Mutation or deletion of the genes responsible for antagonizing PKRshould prevent viral replication in cells in which the PKR activity isnormal (i.e. normal cells). However, if infected cells are unable toactivate the antiviral response mediated through PKR (i.e., Ras-mediatedtumor cells), then these mutant viruses should replicate unheeded andcause cell death. Therefore, these mutant viruses can replicatepreferentially in Ras-transformed cells where it is determined that PKRis unable to function.

In view of the drawbacks associated with the current means for treatingneoplastic growth, the need still exists for improved methods for thetreatment of most types of cancers.

SUMMARY OF THE INVENTION

This invention is directed to a method for treating a Ras-mediated cellproliferative disorder in a mammal, comprising administering toproliferating cells in a mammal having a Ras-activated pathway aneffective amount of one or more viruses selected from the groupconsisting of modified adenovirus, modified HSV, modified vaccinia virusand modified parapoxvirus orf virus under conditions which result insubstantial lysis of the proliferating cells.

The virus is attenuated or modified such that modified adenoviruscomprises a mutant gene encoding VAI RNA, the modified HSV comprises amutation in the gene _(γ1)34.5, the modified vaccinia virus comprises amutant gene selected from the group consisting of E3L and K3L, and themodified parapoxvirus orf virus comprises a mutation in the OV20.0Lgene.

The virus may be modified such that the virion is packaged in a liposomeor micelle, or the proteins of the outer capsid have been mutated. Thevirus can be administered in a single dose or in multiple doses. Thecell proliferative disorder may be a neoplasm. Both solid andhematopoietic neoplasms can be targeted.

Also provided is a method of treating a neoplasm having an activatedRas-pathway in a human, comprising administering to the neoplasm aneffective amount of virus selected from the group consisting of modifiedadenovirus, modified HSV, modified vaccinia virus and modifiedparapoxvirus orf virus, to result in substantial oncolysis of theneoplastic cells.

The virus may be administered by injection into or near a solidneoplasm.

Also provided is a method of inhibiting metastasis of a neoplasm havingan activated Ras-pathway in a mammal, comprising administering to theneoplastic cells in a mammal a virus selected from the group consistingof modified adenovirus, modified HSV, modified vaccinia virus andmodified parapoxvirus orf virus, in an amount sufficient to result insubstantial lysis of the neoplasm.

Also provided is a method of treating a neoplasm suspected of having anactivated Ras-pathway in a mammal, comprising surgical removal of thesubstantially all of the neoplasm and administration of a virus selectedfrom the group consisting of modified adenovirus, modified HSV, modifiedvaccinia virus and modified parapoxvirus orf virus, to the surgical sitein an amount sufficient to result in substantial oncolysis of anyremaining neoplasm.

Also provided is a pharmaceutical composition comprising a virusselected from the group consisting of modified adenovirus, modified HSV,modified vaccinia virus and modified parapoxvirus orf virus, achemotherapeutic agent and a pharmaceutically acceptable excipient.

Also provided is a pharmaceutical composition comprising a virusselected from the group consisting of modified adenovirus, modified HSV,modified vaccinia virus and modified parapoxvirus orf virus, and apharmaceutically acceptable excipient.

Further, this invention includes a kit comprising a pharmaceuticalcomposition comprising a virus selected from the group consisting ofmodified adenovirus, modified HSV, modified vaccinia virus and modifiedparapoxvirus orf virus, and a chemotherapeutic agent.

Additionally, this invention provides a kit comprising a pharmaceuticalcomposition comprising a virus selected from the group consisting ofmodified adenovirus, modified HSV, modified vaccinia virus and modifiedparapoxvirus orf virus and an anti-antivirus antibody.

Also provided is a method for treating a population of cells comprisinga neoplasm suspected of having an activated Ras-pathway in vitrocomprising administering to said population of cells in vitro a virusselected from the group consisting of modified adenovirus, modified HSV,modified vaccinia virus and modified parapoxvirus orf virus in an amountsufficient to result in substantial lysis of the neoplasm.

The invention is also directed to methods of treating a Ras-mediatedproliferative disorder in a mammal, by immunosuppressing,immunoinhibiting or otherwise rendering the mammal immunodeficient and,concurrently or subsequently, administering a virus selected from thegroup consisting of modified adenovirus, modified HSV, modified vacciniavirus and modified parapoxvirus orf virus in an amount sufficient toresult in substantial lysis of the neoplasm. In particular, it isdirected to method for treating a Ras-mediated proliferative disorder ina mammal, by

-   -   a) performing a step selected from the group consisting of:        -   i) administering to the proliferating cells in said mammal            an effective amount of an immune suppressive agent;        -   ii) removing B-cells or T-cells from said mammal;        -   iii) removing anti-virus antibodies from said mammal;        -   iv) removing antibodies from said mammal;        -   v) administering anti-antivirus antibodies to said mammal;            and        -   vi) suppressing the immune system of the mammal; and    -   b) administering to the proliferating cells in said mammal an        effective amount of one or more viruses selected from the group        consisting of modified adenovirus, modified HSV, modified        vaccinia virus and modified parapoxvirus orf virus under        conditions which result in substantial lysis of the        proliferating cells.

The methods and pharmaceutical compositions of the invention provide aneffective means to treat neoplasia having an activated Ras-pathway,without the side effects associated with other forms of cancer therapy.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying FIGURE.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a depiction of the molecular basis of VAI defective adenovirusoncolysis.

DETAILED DESCRIPTION OF THE INVENTION

The invention pertains to methods of treating a Ras-mediatedproliferative disorder in a mammal, by administering a virus selectedfrom the group consisting of modified adenovirus, modified HSV, modifiedvaccinia virus and modified parapoxvirus orf virus, to the proliferatingcells.

DEFINITIONS

The following terms used herein are defied as follows:

“Adenovirus” is a double stranded DNA virus of about 3.6 kilobases. Inhumans, adenoviruses can replicate and cause disease in the eye and inthe respiratory, gastrointestinal and urinary tracts. About one-third ofthe 47 known human serotypes are responsible for most cases of humanadenovirus disease.²⁸ The adenovirus encodes several gene products thatcounter antiviral host defense mechanisms. The virus-associated RNA (VAIRNA or VA RNA₁) of the adenovirus are small, structured RNAs thataccumulate in high concentrations in the cytoplasm at late time afteradenovirus infection. These VAI RNA bind to the to the double strandedRNA (dsRNA) binding motifs of PKR and block the dsRNA-dependentactivation of PKR by autophosphorylation. Thus, PKR is not able tofunction and the virus can replicate within the cell. The overproductionof virons eventually leads to cell death. The attenuated or modifiedadenovirus is unable to replicate in cells which do not have anactivated Ras-pathway. However, attenuated or modified adenovirus canreplicate in cells with an activated Ras-pathway.

The term “attenuated adenovirus” or “modified adenovirus” means that thegene product or products which prevent the activation of PKR arelacking, inhibited or mutated such that PKR activation is not blocked.Preferably, the VAI RNA's are not transcribed. Such attenuated ormodified adenovirus would not be able to replicate in normal cells thatdo not have an activated Ras-pathway, however, it would be able toinfect and replicate in cells having an activated Ras-pathway.

“Herpes simplex virus” (HSV) refers to herpes simplex virus-1 (HSV-1) orherpes simplex virus-2 (HSV-2). HSV gene _(γ1)34.5 encodes the geneproduct infected-cell protein 34.5 (ICP34.5) that can prevent theantiviral effects exerted by PKR. ICP34.5 has a unique mechanism ofpreventing PKR activity by interacting with protein phosphatase 1 andredirecting it activity to dephosphorylate eIF-2α.²⁹ In cells infectedwith either wild-type or the genetically engineered virus from which the_(γ1)34.5 genes were deleted, eIF-2α is phosphorylated and proteinsynthesis is turned off in cells infected with _(γ1)34.5 minus virus. Itwould be expected that the _(γ1)34.5 minus virus would be replicationcompetent in cells with an activated Ras pathway in which the activityof ICP34.5 would be redundant. HSV is unable to replicate in cells whichdo not have an activated Ras-pathway. Thus, HSV can replicate in cellswhich have an activated Ras-pathway.

The term “attenuated HSV” or “modified HSV” means that the gene productor products which prevent the activation of PKR are lacking, inhibitedor mutated such that PKR activation is not blocked. Preferably, the HSVgene _(γ1)34.5 is not transcribed. Such attenuated or modified HSV wouldnot be able to replicate in normal cells that do not have an activatedRas-pathway, however, it would be able to infect and replicate in cellshaving an activated Ras-pathway.

“Parapoxvirus Orf Virus” is a poxvirus. It is a virus that induces acutecutaneous lesions in different mammalian species, including humans.Parpoxvirus orf virus naturally infects sheep, goats and humans throughbroken or damaged skin, replicates in regenerating epidermal cells andinduces pustular leasions that turns to scabs.³⁰ The parapoxvirus orfvirus encodes the gene OV20.0L that is involved in blocking PKRactivity.³⁰ The parapoxvirus orf virus is unable to replicate in cellswhich do not have an activated Ras-pathway. Thus, the parapoxvirus orfvirus replicate in cells which have an activated Ras-pathway.

The term “attenuated parapoxvirus orf virus” or “modified parapoxvirusorf virus” means that the gene product or products which prevent theactivation of PKR are lacking, inhibited or mutated such that PKRactivation is not blocked. Preferably, the gene OV20.0L is nottranscribed. Such attenuated or modified parapoxvirus orf virus wouldnot be able to replicate in normal cells that do not have an activatedRas-pathway, however, it would be able to infect and replicate in cellshaving an activated Ras-pathway.

“Vaccinia virus” refers to the virus of the orthopoxvirus genus thatinfects humans and produces localized lesions.²⁸ Vaccinia virus encodestwo genes that play a role in the down regulation of PKR activitythrough two entirely different mechanisms. E3L gene encodes two proteinsof 20 and 25 kDa that are expressed early in infection and have dsRNAbinding activity that can inhibit PKR activity. Deletion or disruptionof the E3L gene creates permissive viral replication in cells having anactivated Ras pathway. The K3L gene of vaccinia virus encodes pK3, apseudosubstrate of PKR.

Deletion of residues which disrupt E3 function to inhibit the dsRNAbinding. Additionally, since the amino terminal region of E3 proteininteracts with the carboxy-terminal region domain of PKR, deletion orpoint mutation of this domain prevents anti-PKR function. Chang et al.,PNAS 89:4825-4829 (1992); Chang et al., Virol. 194:537-547 (1993); Changet al. J. Virol. 69:6605-6608 (1995); Sharp et al. Virol. 250:302-315(1998); and Romano et al., Molecular and Cellular Bio., 18(12):7304-7316(1998). The K3L gene of vaccinia virus encodes pK3, a pseudosubstrate ofPKR. There is a loss-of-function mutation within K3L. By eithertruncating or by placing point mutations within the C-terminal portionof K3L protein, homologous to residues 79 to 83 in eIF-2α abolish PKRinhibitory activity.³¹

The term “attenuated vaccinia virus” or “modified vaccinia virus” meansthat the gene product or products which prevent the activation of PKRare lacking, inhibited or mutated such that PKR activation is notblocked. Preferably, the E3L gene and/or the K3L gene is nottranscribed. Such attenuated or modified vaccinia virus would not beable to replicate in normal cells that do not have an activatedRas-pathway, however, it would be able to infect and replicate in cellshaving an activated Ras-pathway.

A “proliferative disorder” is any cellular disorder in which the cellsproliferate more rapidly than normal tissue growth. Thus a“proliferating cell” is a cell that is proliferating more rapidly thannormal cells. The proliferative disorder, includes but is not limited toneoplasms. A “neoplasm” is an abnormal tissue growth, generally forminga distinct mass, that grows by cellular proliferation more rapidly thannormal tissue growth. Neoplasms show partial or total lack of structuralorganization and functional coordination with normal tissue. These canbe broadly classified into three major types. Malignant neoplasmsarising from epithelial structures are called carcinomas, malignantneoplasms that originate from connective tissues such as muscle,cartilage, fat or bone are called sarcomas and malignant tumorsaffecting hematopoetic structures (structures pertaining to theformation of blood cells) including components of the immune system, arecalled leukemias and lymphomas. A tumor is the neoplastic growth of thedisease cancer. As used herein, a neoplasm, also referred to as a“tumor”, is intended to encompass hematopoietic neoplasms as well assolid neoplasms. Other proliferative disorders include, but are notlimited to neurofibromatosis.

“Administration to a proliferating cell or neoplasm” indicates that thevirus is administered in a manner so that it contacts the proliferatingcells or cells of the neoplasm (also referred to herein as “neoplasticcells”).

A “mammal suspected of having a proliferative disorder” means that themammal may have a proliferative disorder or tumor or has been diagnosedwith a proliferative disorder or tumor or has been previously diagnosedwith a proliferative disorder or tumor, the tumor or substantially allof the tumor has been surgically removed and the mammal is suspected ofharboring some residual tumor cells.

“Viral infection” or “virus infection” as used herein refers toinfection by one or more of adenovirus, HSV, parapoxvirus orf virus, orvaccinia virus.

“Resistance” of cells to viral infection indicates that infection of thecells with the virus does not result in significant viral production oryield. Without being limited to any theory, resistance to viralinfection is believed to be found at the level of gene translation,rather than at early transcription. While viral transcripts areproduced, viral proteins are not expressed. It is thought that viralgene transcription in resistant cells correlated with phosphorylation ofan approximately 65 kDa cell protein, determined to be double-strandedRNA-activated protein kinase (PKR), that was not observed in transformedcells. Phosphorylation of PKR lead to inhibition of translation.

The term “substantial lysis” means at least 10% of the proliferatingcells are lysed, more preferably of at least 50% and most preferably ofat least 75% of the cells are lysed. The percentage of lysis can bedetermined for tumor cells by measuring the reduction in the size of thetumor in the mammal or the lysis of the tumor cells in vitro.

“Anti-virus antibody” refers to an antibody which binds to a particularvirus. For example, an anti-virus antibody may be an anti-adenovirusantibody, an anti-HSV antibody, an anti-vaccinia virus antibody or ananti-parapoxvirus orf virus antibody. The particular anti-virus antibodyselected for use in the methods of this invention will correspond to thevirus which is administered to the patient. For example, an anti-HSVantibody would be used in the method where a modified HSV isadministered.

“Anti-antivirus antibodies,” are antibodies directed against anti-virusantibodies. Anti-antivirus antibodies used in this invention areselected from anti-antiadenovirus antibodies, anti-antiHSV antibodies,anti-antivaccinia virus antibodies and anti-antiparapoxvirus orf virusantibodies. Such antibodies can be made by methods known in the art. Seefor example “Antibodies: A laboratory manual” E. Harlow and D. Lane,Cold Spring Harbor Laboratory (1988).

“IgG antibodies” refers to immunoglobulin G antibodies. IgG, the mostabundant type of antibody, carries the major burden of neutralizingbacterial toxins and binding to microorganisms to enhance theirphagocytosis.

“Humanized antibodies” refers to antibody molecules in which the aminoacid sequence in the non-antigen binding regions has been altered sothat the antibody more closely resembles a human antibody, and stillretains its original binding ability.

The terms “immunosuppressant” or “immune suppressive agent” includeconventional immunosuppressants, immunoinhibitors, antibodies, andconditions such as radiation therapy or HIV infection which result incompromise of the immune system.

“B-cells” refers to B-lymphocytes. There are two major subpopulations ofB lymphocytes, B-1 and B-2 cells. B-1 cells are self-renewing andfrequently secrete high levels of antibody which bind to a range ofantigens (polyspecificity) with a relatively low affinity. The majorityof B cells, B-2 cells, are directly generated from precursors in thebone marrow and secrete highly specific antibody.

“T-cells” refers to T-lymphocytes. T-cells differentiate within thethymus gland and are specialized to operate against cells bearingintracellular organisms. T-cells only recognize antigen when it is onthe surface of a body cell.

It is believed that the virus uses the host cell's Ras pathway machineryto downregulate PKR and thus reproduce. FIG. 1 depicts the usurpation ofthe host cell Ras signalling pathway by adenovirus. As shown in FIG. 1,in both untransformed and Ras-activated cells, wild-type adenovirus(denoted with +) and VAI defective adenovirus (open circle) are bothable to bind, internalize and undergo early transcription in a normalfashion.

During transcription, wild-type adenovirus (panel #1) is able totranscribe VAI RNAs that can bind to PKR without activating it. BecausePKR is unable to displace these short, double stranded RNAs (dsRNAs),PKR is unable to interact with subsequent longer transcripts andautophosphorylate. Thus, the virus is able to replicate and produceprogeny virus.

When attempting to replicate in untransformed cells (panel #2), modifiedadenovirus is unable to produce the VAI RNAs which bind to PKR. Thus,PKR can interact with longer viral transcripts that are capable ofcausing autophosphorylation and activate PKR. The activated PKR is thenable to phosphorylate the translation initiation factor eIF-2α and blocktranslation of viral genes that lead to abortive viral replication.

Panel #3 shows the modified adenovirus infecting a Ras-activated cancercell where the outcome is different from the outcome described in panels#1 and #2. In the Ras-transformed cells, it has been observed that PKRis unable to undergo phosphorylation or that phosphorylation is rapidlyreversed by an element of the activated Ras pathway. The result in theRas-activated cells is that the modified form of the adenovirus is ableto translate its viral genes and complete replication without thetranscription of the VAI RNAs. The surprising result in these cells isoncolysis.

As is known in the art, the implantation of human tumor cells into SCIDmice is recognized as a well known model system for testing theeffectiveness of various anti-tumor agents in humans. It has previouslybeen shown that pharmaceuticals effective against human tumors implantedinto SCID mice can be predictive of their effectiveness against the sametumors in humans.

Based upon these discoveries, Applicants have developed methods fortreating cell proliferative disorders in mammals wherein the cells havean activated Ras-pathway. Representative mammals include dogs, cats,sheep, goats, cattle, horses, pigs, non-human primates, and humans. In apreferred embodiment, the mammal is a human.

Methods of the Invention

In the methods of the invention, modified virus is administered toproliferating cells having an activated Ras-pathway in the individualmammal. Representative types of modified virus include adenovirus, HSV,parapoxvirus orf virus, or vaccinia virus which infect humans. In apreferred embodiment, modified adenovirus is used.

The virus may be a recombinant virus from two or more types of viruseswith differing pathogenic phenotypes such that it contains differentantigenic determinants thereby reducing or preventing an immune responseby a mammal previously exposed to a virus subtype. Such recombinantvirions can be generated by co-infection of mammalian cells withdifferent subtypes of virus with the resulting resorting andincorporation of different subtype coat proteins into the resultingvirion capsids.

The virus may be modified by incorporation of mutated coat proteins intothe virion outer capsid. The proteins may be mutated by replacement,insertion or deletion. “Replacement” includes the insertion of differentamino acids in place of the native amino acids. “Insertions” include theinsertion of additional amino acid residues into the protein at one ormore locations. “Deletions” include deletions of one or more amino acidresidues in the protein. Such mutations may be generated by methodsknown in the art. For example, oligonucleotide site directed mutagenesisof the gene encoding for one of the coat proteins could result in thegeneration of the desired mutant coat protein. Expression of the mutatedprotein in virus infected mammalian cells in vitro such as COS1 cellswill result in the incorporation of the mutated protein into the virusvirion particle

The virus is preferably a virus modified to reduce or eliminate animmune reaction to the virus. Such modified virus are termed“immunoprotected virus”. Such modifications could include packaging ofthe virus in a liposome, a micelle or other vehicle to mask the virusfrom the mammals immune system.

At least some of the cells of the proliferative disorder have a mutationin which the Ras gene (or an element of the Ras signaling pathway) isactivated, either directly (e.g., by an activating mutation in Ras) orindirectly (e.g., by activation of an upstream element in the Raspathway). Activation of an upstream element in the Ras pathway includes,for example, transformation with epidermal growth factor receptor (EGFR)or Sos. A proliferative disorder that results, at least in part, by theactivation of Ras, an upstream element of Ras, or an element in the Rassignalling pathway is referred to herein as a “Ras-mediatedproliferative disorder”.

One neoplasm that is particularly susceptible to treatment by themethods of the invention is pancreatic cancer, because of the prevalenceof Ras-mediated neoplasms associated with pancreatic cancer. Otherneoplasms that are particularly susceptible to treatment by the methodsof the invention include breast cancer, central nervous system cancer(e.g., neuroblastoma and glioblastoma), peripheral nervous systemcancer, lung cancer, prostate cancer, colorectal cancer, thyroid cancer,renal cancer, adrenal cancer, liver cancer, lymphoma and leukemia. Oneproliferative disorder that is particularly susceptible to treatment bythe methods of this invention include neurofibromatosis 1 because of theactivation of the Ras pathway.

The virus is administered to a proliferating cell or neoplasm in amanner so that it contacts the proliferating cells or cells of theneoplasm or neoplastic cells. The route by which the virus isadministered, as well as the formulation, carrier or vehicle, willdepend on the location as well as the type of the neoplasm. A widevariety of administration routes can be employed. For example, for asolid neoplasm that is accessible, the virus can be administered byinjection directly to the neoplasm. For a hematopoietic neoplasm, forexample, the virus can be administered intravenously or intravascularly.For neoplasms that are not easily accessible within the body, such asmetastases or brain tumors, the virus is administered in a manner suchthat it can be transported systemically through the body of the mammaland thereby reach the neoplasm (e.g., intrathecally, intravenously orintramuscularly).

Alternatively, the virus can be administered directly to a single solidneoplasm, where it then is carried systemically through the body tometastases. The virus can also be administered subcutaneously,intraperitoneally, topically (e.g., for melanoma), orally (e.g., fororal or esophageal neoplasm), rectally (e.g., for colorectal neoplasm),vaginally (e.g., for cervical or vaginal neoplasm), nasally or byinhalation spray (e.g., for lung neoplasm).

Virus can be administered systemically to mammals which are immunecompromised or which have not developed immunity to the virus epitopes.In such cases, virus administered systemically, i.e. by intravenousinjection, will contact the proliferating cells resulting in lysis ofthe cells.

Immunocompetent mammals previously exposed to a particular virus, suchas modified adenovirus, modified HSV, modified vaccinia virus andmodified parapoxvirus orf virus, may have developed humoral and/orcellular immunity to that virus. Nevertheless, it is contemplated thatdirect injection of the virus into a solid tumor in immunocompetentmammals will result in the lysis of the neoplastic cells.

When the virus is administered systemically to immunocompetent mammals,the mammals may produce an immune response to the virus. Such an immuneresponse may be avoided if the virus is of a subtype to which the mammalhas not developed immunity, or the virus has been modified as previouslydescribed herein such that it is immunoprotected, for example, byprotease digestion of the outer capsid or packaging in a micelle.

It is contemplated that the virus may be administered to immunocompetentmammals immunized against the virus in conjunction with theadministration of anti-antivirus antibodies. Such anti-antivirusantibodies may be administered prior to, at the same time or shortlyafter the administration of the virus. Preferably an effective amount ofthe anti-antivirus antibodies are administered in sufficient time toreduce or eliminate an immune response by the mammal to the administeredvirus.

Alternatively, it is contemplated that the immunocompetency of themammal against the virus may be suppressed either by the prior orco-administration of pharmaceuticals known in the art to suppress theimmune system in general (Cuff et al., “Enteric reovirus infection as aprobe to study immunotoxicity of the gastrointestinal tract”Toxicological Sciences 42(2):99-108 (1998)) or alternatively theadministration of such immunoinhibitors as anti-antivirus antibodies.

The humoral immunity of the mammal against the virus may also betemporarily reduced or suppressed by plasmaphoresis of the mammals bloodto remove the anti-virus antibodies. The anti-virus antibodies removedby this process correspond to the virus selected for administration tothe patient. For example, if a modified parapox orf virus is selectedfor administration, then the anti-parapox orf virus antibodies will beremoved. The humoral immunity of the mammal against the virus mayadditionally be temporarily reduced or suppressed by the intravenousadministration of non-specific immunoglobulin to the mammal.

Other agents are known to have immunosuppressant properties as well(see, e.g., Goodman and Gilman, 7′' Edition, page 1242, the disclosureof which is incorporated herein by reference). Such immunoinhibitorsalso include anti-antivirus antibodies, which are antibodies directedagainst anti-virus antibodies. Anti-antivirus antibodies used in thisinvention are selected from anti-antiadenovirus antibodies, anti-antiHSVantibodies, anti-antivaccinia virus antibodies and anti-antiparapoxvirusorf virus antibodies. Such antibodies can be made by methods known inthe art. See for example “Antibodies: A laboratory manual” E. Harlow andD. Lane; Cold Spring Harbor Laboratory (1988).

Such anti-antivirus antibodies may be administered prior to, at the sametime or shortly after the administration of the virus. Preferably aneffective amount of the anti-antivirus antibodies are administered insufficient time to reduce or eliminate an immune response by the mammalto the administered virus.

In yet other methods of the invention, a virus selected from the groupconsisting of modified adenovirus, modified HSV, modified vaccinia virusand modified parapoxvirus orf virus is administered to Ras-mediatedproliferating cells in the individual mammal. In one embodiment of thisinvention a course of this therapy is administered one or more times.Following the first administration of virus therapy particular immuneconstituents that may interfere with subsequent administrations of virusare removed from the patient. These immune constituents include B cells,T cells, antibodies, and the like.

Removal of either the B cell or T cell population can be accomplished byseveral methods. In one method, the blood may be filtered andheme-dialysis may be performed. Another method is the filtration of theblood coupled with extra corporeal compounds that can remove the cellpopulations, for example, with immobilized antibodies that recognizespecific receptors on the cell population which is to be remove. Yetanother method for removal of a cell population is by immunesuppression. This can be done by first line radiation therapy or bycyclic steroids such as cyclosporin.

Selective removal of anti-virus antibodies can also prevent thepatient's immune system from removing therapeutically administeredvirus. Preventing antibody interaction with the administered virus mayalso assist systemic treatment strategies. Antibodies can be removed byseveral methods, including heme-dialysis and passing the blood overimmobilized virus (selective antibody removal); by removal of all IgGantibodies by heme-dialysis and passing the blood over immobilizedprotein A (commercially available as PROSORBA, Cypress Bioscience, SanDiego, Calif.); or by administration of humanized anti-idiotypicantibodies, where the idiotype is against the virus to be administered(e.g., a virus selected from the group consisting of modifiedadenovirus, modified HSV, modified vaccinia virus and modifiedparapoxvirus orf virus).

Another method of this invention is to allow virus to act systemicallywithout impairing normal immune function by masking or impairing immunerecognition of virus. To prevent the patient's immune system fromrecognizing the administered virus, the virus may be coated withnon-virotoxic humanized antibodies, such as coating with the F_(ab)portion of the antibody, or coated in a micelle.

Additionally, the virus may be treated with chymotrypsin to yield aninfectious subviral particle (ISVP). An ISVP may be used either alone orin combination with whole virus to provide an agent that is eitherpoorly recognized has not been previously prevented by the patient'simmune system.

Another embodiment of this invention includes the removal of virus fromthe patient following administration. Since this method may be used onpatients that are either immune suppressed or immune incompetent, it maybe of importance to remove virus from the blood stream following thecourse of treatment. Virus may be removed by affinity chromatographyusing extra corporeal anti-virus antibodies associated with hemedialysis, B-cell proliferative agents, or adjuvants to stimulate immuneresponse against the virus such as UV inactivated virus or Freund'sadjuvant.

Pharmaceutical Compositions

This invention also includes pharmaceutical compositions which contain,as the active ingredient, one or more of the viruses associated with“pharmaceutically acceptable carriers or excipients.” This inventionalso includes pharmaceutical compositions which contain, as the activeingredient, one or more immunosuppressants or immunoinhibitors and oneor more of the viruses associated with “pharmaceutically acceptablecarriers or excipients.”

In making the compositions of this invention, the active ingredient(s),e.g., the virus and/or immunosuppressant or immunoinhibitor, are usuallymixed with an excipient, diluted by an excipient or enclosed within sucha carrier which can be in the form of a capsule, sachet, paper or othercontainer.

When the pharmaceutically acceptable excipient serves as a diluent, itcan be a solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, sterile water, syrup, and methylcellulose. The formulations can additionally include: lubricating agentssuch as talc, magnesium stearate, and mineral oil; wetting agents;emulsifying and suspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

For preparing solid compositions such as tablets, the principal activeingredient/virus is mixed with a pharmaceutical excipient to form asolid preformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the novel compositions of the presentinvention may be incorporated for administration orally or by injectioninclude aqueous solutions, suitably flavored syrups, aqueous or oilsuspensions, and flavored emulsions with edible oils such as corn oil,cottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein. Preferably the compositions are administered by the oral ornasal respiratory route for local or systemic effect. Compositions inpreferably pharmaceutically acceptable solvents may be nebulized by useof inert gases. Nebulized solutions may be inhaled directly from thenebulizing device or the nebulizing device may be attached to a facemask tent, or intermittent positive pressure breathing machine.Solution, suspension, or powder compositions may be administered,preferably orally or nasally, from devices which deliver the formulationin an appropriate manner.

Another preferred formulation employed in the methods of the presentinvention employs transdermal delivery devices (“patches”). Suchtransdermal patches may be used to provide continuous or discontinuousinfusion of the virus of the present invention in controlled amounts.The construction and use of transdermal patches for the delivery ofpharmaceutical agents is well known in the art. See, for example, U.S.Pat. No. 5,023,252, herein incorporated by reference. Such patches maybe constructed for continuous, pulsatile, or on demand delivery ofpharmaceutical agents.

Other suitable formulations for use in the present invention can befound in Remington's Pharmaceutical Sciences.

Kits of Parts

The virus or the pharmaceutical composition comprising the virus may bepackaged into convenient kits providing the necessary materials packagedinto suitable containers. It is contemplated the kits may also includechemotherapeutic agents and/or anti-antivirus antibody.

The immunosuppressant or immunoinhibitor and virus or the pharmaceuticalcomposition comprising the immunosuppressant or immunoinhibitor andvirus may be packaged into convenient kits providing the necessarymaterials packaged into suitable containers. It is contemplated the kitsmay also include chemotherapeutic agent.

Administration of Virus

The virus is administered in an amount that is sufficient to treat theproliferative disorder (e.g., an “effective amount”). A proliferativedisorder is “treated” when administration of virus to the proliferatingcells effects lysis of the proliferating cells. This may result in areduction in size of the neoplasm, or in a complete elimination of theneoplasm. The reduction in size of the neoplasm, or elimination of theneoplasm, is generally caused by lysis of neoplastic cells (“oncolysis”)by the virus.

Preferably, the effective amount is that amount able to inhibit tumorcell growth. Preferably the effective amount is from about 1.0 pfu/kgbody weight to about 10¹⁵ pfu/kg body weight, more preferably from about10² pfu/kg body weight to about 10¹³ pfu/kg body weight. For example,for treatment of a human, approximately 10² to 10¹⁷ plaque forming units(PFU) of virus can be used, depending on the type, size and number oftumors present. The effective amount will be determined on an individualbasis and may be based, at least in part, on consideration of the typeof virus; the chosen route of administration; the individual's size,age, gender; the severity of the patient's symptoms; the size and othercharacteristics of the neoplasm; and the like. The course of therapy maylast from several days to several months or until diminution of thedisease is achieved.

The virus can be administered in a single dose, or multiple doses (i.e.,more than one dose). The multiple doses can be administeredconcurrently, or consecutively (e.g., over a period of days or weeks).The virus can also be administered to more than one neoplasm in the sameindividual.

The compositions are preferably formulated in a unit dosage form, eachdosage containing from about 10² pfus to about 10¹³ pfus of the virus.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of virus calculated to producethe desired therapeutic effect, in association with a suitablepharmaceutical excipient.

It has been found that the virus is effective for the treatment of solidneoplasms in immunocompetent mammals. Administration of unmodified virusdirectly to the neoplasm results in oncolysis of the neoplastic cellsand reduction in the size of the tumor.

It is contemplated that the virus may be administered in conjunctionwith surgery or removal of the neoplasm. Therefore, provided herewithare methods for the treatment of a solid neoplasm comprising surgicalremoval of the neoplasm and administration of a virus at or near to thesite of the neoplasm.

It is contemplated that the virus may be administered in conjunctionwith or in addition to radiation therapy.

It is further contemplated that the virus of the present invention maybe administered in conjunction with or in addition to known anti-cancercompounds or chemotherapeutic agents. Chemotherapeutic agents arecompounds which may inhibit the growth of tumors. Such agents, include,but are not limited to, 5-fluorouracil, mitomycin C, methotrexate,hydroxyurea, cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins(Epirubicin and Doxurubicin), antibodies to receptors, such asherceptin, etopside, pregnasome, platinum compounds such as carboplatinand cisplatin, taxanes such as taxol and taxotere, hormone therapiessuch as tamoxifen and anti-estrogens, interferons, aromatase inhibitors,progestational agents and LHRH analogs. In one embodiment of theinvention, a method is provided for reducing the growth of metastastictumors in a mammal comprising administering an effective amount of avirus to the mammal.

Administration of Virus with Immunosuppressant or Immunoinhibitor

The immunosuppressant or immunoinhibitor is administered in anappropriate amount and using an appropriate schedule of administrationsufficient to result in immunosuppression or immunoinhibition of themammal's immune system. Such amounts and schedules are well known tothose of skill in the art.

The immunosuppressant or immunoinhibitor and virus can be administeredin a single dose, or multiple doses (i.e., more than one dose). Themultiple doses can be administered concurrently, or consecutively (e.g.,over a period of days or weeks). The virus can also be administered tomore than one neoplasm in the same individual.

The compositions are preferably formulated in a unit dosage form, eachdosage containing an appropriate amount of immunosuppressant orimmunoinhibitor and from about 10² pfus to about 10¹³ pfus of the virus.The term “unit dosage forms” refers to physically discrete unitssuitable as unitary dosages for human subjects and other mammals, eachunit containing a predetermined quantity of virus calculated to producethe desired therapeutic effect, in association with a suitablepharmaceutical excipient.

As mentioned above, it has been found that the virus is effective forthe treatment of solid neoplasms in immunocompetent mammals.Administration of unmodified virus directly to the neoplasm results inoncolysis of the neoplastic cells and reduction in the size of the tumorin immunocompetent animals. When animals are rendered immunosuppressedor immunodeficient in some way, systemic administration of virus will bemore effective in producing oncolysis.

It is contemplated that the virus may be administered in conjunctionwith or in addition to radiation therapy which renders the mammalimmunosuppressed. It is further contemplated that the virus andimmunosuppressant or immunoinhibitor may be administered in conjunctionwith or in addition to known anti-cancer compounds or chemotherapeuticagents. Chemotherapeutic agents are compounds which may inhibit thegrowth of tumors. Such agents, include, but are not limited to,5-fluorouracil, mitomycin C, methotrexate, hydroxyurea,cyclophosphamide, dacarbazine, mitoxantrone, anthracyclins (Epirubicinand Doxurubicin), antibodies to receptors, such as herceptin, etopside,pregnasome, platinum compounds such as carboplatin and cisplatin,taxanes such as taxol and taxotere, hormone therapies such as tamoxifenand anti-estrogens, interferons, aromatase inhibitors, progestationalagents and LHRH analogs.

The virus and immunosuppressants of the present invention arecontemplated to reduce the growth of tumors that are metastatic. In anembodiment of the invention, a method is provided for reducing thegrowth of metastatic tumors in a mammal comprising administering aneffective amount of a virus to the immunosuppressed mammal.

It is contemplated that the selected virus may be administered toimmunocompetent mammals immunized against the selected virus inconjunction with the administration of immunosuppressants and/orimmunoinhibitors. For example, if a modified vaccinia virus is selectedthen the immunocompetent mammal is immunized against vaccinia virus.Such immunosuppressants and immunoinhibitors are known to those of skillin the art and include such agents as cyclosporin, rapamycin,tacrolimus, mycophenolic acid, azathioprine and their analogs, and thelike.

Utility

The viruses of the present invention may be used for a variety ofpurposes. They may be used in methods for treating Ras-mediatedproliferative disorders in a mammal. The virus may be used to reduce oreliminate neoplasms. They may be used in methods for treatingmetastases. They may be used in conjunction with known treatments forcancer including surgery, chemotherapy and radiation.

In order to further illustrate the present invention and advantagesthereof, the following specific examples are given but are not meant tolimit the scope of the claims in any way.

EXAMPLES

In the examples below, all temperatures are in degrees Celsius (unlessotherwise indicated) and all percentages are weight percentages (alsounless otherwise indicated).

In the examples below, the following abbreviations have the followingmeanings. If an abbreviation is not defined, it has its generallyaccepted meaning:

-   -   μM=micromolar    -   mM=millimolar    -   M=molar    -   ml=milliliter    -   μl=microliter    -   mg=milligram    -   μg=microgram    -   DNA=deoxyribonucleic acid    -   RNA=ribonucleic acid    -   PAGE=polyacrylamide gel electrophoresis    -   rpm=revolutions per minute    -   FBS=fetal bovine serum    -   DTT=dithiothrietol    -   SDS=sodium dodecyl sulfate    -   PBS=phosphate buffered saline    -   DMEM=Dulbecco's modified Eagle's medium    -   α-MEM=α-modified Eagle's medium    -   β-ME=β-mercaptoethanol    -   MOI=multiplicity of infection    -   PFU=plaque forming units    -   MAPK=MAP kinase    -   phosph-MAPK=phosphorylated-MAP kinase    -   HRP=horseradish-peroxidase    -   PKR=double-stranded RNA activated protein kinase    -   RT-PCR=reverse transcriptase-polymerase chain reaction    -   GAPDH=glyceraldehyde-3-phosphate dehydrogenase    -   EGFR=epidermal growth factor receptors    -   MEK kinase=mitogen-activated extracellular signal-regulated        kinase    -   DMSO=dimethylsulfoxide    -   SCID=severe combined immunodeficiency        General Methods        Cells and Virus

293 cells (human embryonic kidney (HEK) cells (available from ATCC)) aregrown as monolayers in Dulbecco's modified Eagle's medium (DMEM, GIBCOLaboratories) supplemented with 10% newborn calf serum (NC) and assuspension cultures in minimal essential medium (SMEM, GIBCOLaboratories) supplemented with 5% NCS.

VAI mutant adenovirus are propagated in suspension cultures of 293 cellsmaintained in the same medium. Plaque assays are performed on HeLa and293 monolayers in DMEME containing 0.7% agarose, 2% NCS, 2 mML-glutamine, MEM nonessential acids (GIBCO Laboratories), and 25 mMMgCl₂.

Example 1 In Vivo Oncolytic Capability of Adenovirus Against HumanBreast Cancer-Derived Cell Lines

In vivo studies are carried out using human breast carcinoma cells in aSCID mouse model. Female SCID mice are injected with 1×10⁶ human breastcarcinoma MDA-MB468 cells in two subcutaneous sites, overlying both hindflanks. Palpable tumors are evident approximately two to four weeks postinjection. Undiluted adenovirus is injected into the right side tumormass in a volume of 20 μl at a concentration of 1.0×10⁷ PFU/ml.

Example 2 Susceptibility of Additional Human Tumors to AdenovirusOncolysis

Cells and Virus

All cell lines are grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% fetal bovine serum (FBS).

The adenovirus used in these studies is propagated in suspensioncultures of L cells and purified as described above.

Cytopathic Effects of Adenovirus on Cells

Confluent monolayers of cells are infected with adenovirus at amultiplicity of infection (MO1) of approximately 40 plaque forming units(PFU) per cell.

Pictures are taken at 36 hour postinfection for both adenovirus-infectedand mock infected cells.

Immunofluorescent Analysis of Adenovirus Infection

For the immunofluorescent studies the cells are grown on coverslips, andinfected with adenovirus at a multiplicity of infection (MOI) of ˜10PFU/cell or mock-infected as described above. At various timespostinfection, cells are fixed in an ethanol/acetic acid (20/1) mixturefor 5 minutes, then rehydrated by subsequential washes in 75%, 50% and25% ethanol, followed by 4 washes with phosphate-buffered saline (PBS).The fixed and rehydrated cells are then exposed to the primary antibody(rabbit polyclonal anti-adenovirus serum diluted 1/100 in PBS) for 2 hrat room temperature. Following 3 washes with PBS, the cells are exposedto the secondary antibody [goat anti-rabbit IgG (whole molecule)fluorescein isothiocyanate (FITC) conjugate diluted 1/100 in PBScontaining 10% goat serum and 0.005% Evan's Blue counterstain] for 1hour at room temperature. Finally, the fixed and treated cells arewashed 3 more times with PBS, followed by 1 wash with double-distilledwater, dried and mounted on slides in 90% glycerol containing 0.1%phenylenediamine, and viewed with a Zeiss Axiophot microscope mountedwith a Carl Zeiss camera (magnification for all pictures was 200×).

Infection of Cells and Quantitation of Virus

Confluent monolayers of cells grown in 24-well plates are infected withadenovirus at an estimated multiplicity of 10 PFU/cell. After 1 hourincubation at 37° C., the monolayers are washed with warm DMEM-10% FBS,and then incubated in the same medium. At various times postinfection, amixture of NP-40 and sodium deoxycholate is added directly to the mediumon the infected monolayers to final concentrations of 1% and 0.5%,respectively. The lysates are then harvested and virus yields aredetermined by plaque titration on L-929 cells.

Radiolabelling of Adenovirus-Infected Cells and Preparation of Lysates

Confluent monolayers of cells are infected with adenovirus (MOI ˜10PFU/cell). At various times postinfection, the media is replaced withmethionine-free DMEM containing 10% dialyzed PBS and 0.1 mCi/ml[³⁵S]methionine. After further incubation for 1 hour at 37° C., thecells are washed in phosphate-buffered saline (PBS) and lysed in thesame buffer containing 1% Triton X-100, 0.5% sodium deoxycholate and 1mM EDTA. The nuclei are then removed by low speed centrifugation and thesupernatants stored at 70° C. until use.

Immunoprecipitation and SDS-PAGE Analysis

Standard immunoprecipitation of [³⁵S]-labelled adenovirus-infected celllysates with anti-adenovirus serum is done. Immunoprecipitates areanalyzed by discontinuous SDS-PAGE according to the protocol of Laemmli(Laemmli, U.K., (1970) Nature, 227:680-685).

Breast Cancer

The c-erbB-2/neu gene encodes a transmembrane protein with extensivehomology to the EGFR that is overexpressed in 20-30% of patients withbreast cancer (Yu, D. et al. (1996) Oncogene 13:1359). Ras activation,either through point mutations or through augmented signaling cascadeelements upstream of Ras (including the c-erbB-2/neu homologue EGFR)ultimately creates a hospitable environment for virus replication, anarray of cell lines derived from human breast cancers are assayed foradenovirus susceptibility. The cell lines included MDA-MD-435SD (ATCCdeposit HTB-129), MCF-7 (ATCC deposit HTB-22), T-27-D (ATCC depositHTB-133), BT-20 (ATCC deposit HTB-19), HBL-100 (ATCC deposit HTB-124),MDA-MB-468 (ATCC deposit HTB-132), and SKBR-3 (ATCC deposit HTB-30).

Based upon induction of cytopathic effects and viral protein synthesisas measured by radioactive metabolic labeling and immunofluorescence asdescribed above, sensitivity to infection may be determined.

Brain Glioblastoma

Human brain glioblastoma cell lines A-172, U-118, U-178, U-563, U-251,U-87 and U-373 (these cells are a generous gift from Dr. Wee Yong,University of Calgary) are tested to determine the susceptibility toadenovirus infection.

To assess the sensitivity of these cells to adenovirus, cells are grownto 80% confluency and are then challenged with adenovirus at amultiplicity of infection (MO1) of 10. Within a period of 48 hours,widespread cytopathic effects will be seen. To demonstrate further thatthe lysis of these cells is due to replication of adenovirus, the cellsare then pulse-labeled with [³⁵S]methionine for three hour periods atvarious times post-infection and proteins are analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as describedabove.

U-87 cells are also introduced as human tumor xenografts into the hindflank of 10 SCID mice. U-87 cells are grown in Dulbecco's modifiedEagle's medium containing 10% fetal bovine serum, as described above.Cells are harvested, washed, and resuspended in sterile PBS; 2.0×10⁶cells in 100 μl, and are injected subcutaneously at a site overlying thehind flank in five- to eight-week old male SCID mice (Charles River,Canada). Tumor growth is measured twice weekly for a period of fourweeks.

To determine if there is viral spread beyond the tumor mass,immunofluorescent microscopy using antibodies directed against totaladenovirus proteins is conducted, as described above, on paraffinsections of the tumor and adjoining tissue.

Since most tumors are highly vascularized, it is likely that some virusmay enter the blood stream following the lysis of the infected tumorcells. To determine if there is systemic spread of the virus, blood isharvested from the treated and control animals, serially diluted forsubsequent plaque titration, and the concentration of infectious virusin the blood is determined.

The high degree of tumor specificity of the virus, combined withsystemic spread, suggest that adenovirus can replicate in glioblastomatumors remote from the initially infected tumor. SCID mice are implantedbilaterally with U-87 human tumor xenografts on sites overlying eachhind flank of the animals. These tumors are allowed to grow until theymeasure 0.5×0.5 cm. The left-side tumors are then injected with a singledose (1×10⁷ pfu) of adenovirus in treated animals (n=5); control animals(n=7) are mock-treated with UV-inactivated virus. Tumors are againmeasured twice weekly for a period of four weeks.

Pancreatic Carcinoma

Cell lines derived from pancreatic cancer are investigated for theirsusceptibility to adenovirus infection, using processes described above.The cell lines included Capan-1 (ATCC deposit HTB-79), BxPC3 (ATCCdeposit CRL1687), MIAPACA-2 (ATCC deposit CRL-1420), PANC-1 (ATCCdeposit CRL-1469), AsPC-1 (ATCC deposit CRL-1682) and Hs766T (ATCCdeposit HTB-134).

The assays described above may be modified by one skilled in the art totest the susceptibility of cells to other types of virus, such as HSV,vaccinia virus and parapoxvirus orf virus.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims.

1. A method for treating a Ras-mediated cell proliferative disorder in amammal, comprising the steps of: (a) selecting a mammal with aras-mediated cell proliferative disorder comprising cells having anactivated ras-pathway, wherein activation of PKR is blocked in thecells; and (b) administering to the cells an effective amount of atleast one modified vaccinia virus under conditions which result insubstantial lysis of the proliferating cells, wherein the vaccinia viruscomprises a mutant gene selected from the group consisting of E3L andK3L.
 2. The method of claim 1, wherein more than two strains of modifiedvaccinia virus are administered.
 3. The method of claim 1, wherein theRas-mediated proliferative disorder is a neoplasm.
 4. The method ofclaim 1, wherein the Ras-mediated proliferative disorder isneurofibromatosis.
 5. The method of claim 3, wherein the neoplasm is asolid neoplasm.
 6. The method of claim 3, wherein the neoplasm isselected from the group consisting of lung cancer, prostate cancer,colorectal cancer, thyroid cancer, renal cancer, adrenal cancer, livercancer, pancreatic cancer, breast cancer and central and peripheralnervous system cancer.
 7. The method of claim 6, wherein the neoplasm isa central nervous system cancer.
 8. The method of claim 6, wherein theneoplasm is breast cancer.
 9. The method of claim 3, wherein theneoplasm is a hematopoietic neoplasm.
 10. The method of claim 5, whereinthe modified vaccinia virus is administered by injection into or nearthe solid neoplasm.
 11. The method of claim 1, wherein the modifiedvaccinia virus is administered intravenously into the mammal.
 12. Themethod of claim 1, wherein the modified vaccinia virus is administeredintraperitoneally into the mammal.
 13. The method of claim 1 wherein themammal is immunocompetent.
 14. The method of claim 13 wherein themodified vaccinia virus is immunoprotected.
 15. The method of claim 14wherein the modified vaccinia virus is encapsulated in a micelle. 16.The method of claim 13 wherein the modified vaccinia virus isadministered with an effective amount of an anti-antivaccinia virusantibody.
 17. The method of claim 1, wherein the mammal is a human. 18.The method of claim 1, wherein approximately 1 to 10¹⁵ plaque formingunits of modified vaccinia virus/kg body weight are administered. 19.The method of claim 1, wherein the modified vaccinia virus isadministered in a single dose.
 20. The method of claim 1, wherein themodified vaccinia virus is administered in more than one dose.
 21. Themethod of claim 3, wherein the neoplasm is metastatic.
 22. The method ofclaim 1 further comprising the administration of an effective amount ofa chemotherapeutic agent.
 23. The method of claim 1, wherein themodified vaccinia virus is treated with a protease prior toadministration.
 24. A method of treating a neoplasm having an activatedRas-pathway in a human, comprising the steps of: (a) selecting a humanwith a neoplasm comprising cells having an activated Ras-pathway,wherein activation of PKR is blocked in the cells; and (b) administeringto the cells an effective amount of at least one modified vacciniavirus, to result in substantial oncolysis of the cells, wherein thevaccinia virus comprises a mutant gene selected from the groupconsisting of E3L and K3L.
 25. The method of claim 24, wherein theneoplasm is a solid neoplasm and the modified vaccinia virus isadministered by injection into or near the neoplasm.
 26. The method ofclaim 25, wherein the solid neoplasm is pancreatic cancer.
 27. A methodof inhibiting metastasis of a neoplasm having an activated Ras-pathwayin a mammal, comprising the steps of: (a) selecting a mammal with aneoplasm comprising neoplastic cells having an activated Ras-pathway,wherein activation of PKR is blocked in the neoplastic cells; and (b)administering to the neoplastic cells in the mammal at least onemodified vaccinia virus, in an amount sufficient to result insubstantial lysis of the neoplastic cells, wherein the vaccinia viruscomprises a mutant gene selected from the group consisting of E3L andK3L.
 28. The method of claim 27 wherein the mammal is selected from thegroup consisting of dogs, cats, sheep, goats, cattle, horses, pigs,humans and non-human primates.